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102 Science in China: Series D Earth Sciences 2006 Vol.49 Supp.

I 102113
www.scichina.com www.springerlink.com
DOI: 10.1007/s11430-006-8110-z
Contributions of phosphatase and microbial activity to
internal phosphorus loading and their relation to
lake eutrophication
SONG Chunlei
1,2
, CAO Xiuyun
1,2
, LI Jianqiu
1
,

LI Qingman
1
,

CHEN Guoyuan
1,2
& ZHOU Yiyong
1
1. Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China;
2. Graduate School of the Chinese Academy of Sciences, Beijing 100039, China
Correspondence should be addressed to Zhou Yiyong (email: zhouyy@ihb.ac.cn)
Received September 16, 2005; accepted February 6, 2006
Abstract Phosphatase may accelerate the process of lake eutrophication through improving
phosphorus bioavailability. This mechanism was studied in three Chinese eutrophic shallow lakes
(Lake Taihu, Lake Longyang and Lake Lianhua). Phosphatase activity was related to the concentra-
tion of soluble reactive phosphorus (SRP) and chlorophyll a. Stability of dissolved phosphatase in
reverse micelles may be attributed to molecular size, conformation and active residues of the enzyme.
At the site with Microcystis bloomed in Lake Taihu, dissolved phosphatase activity was higher and
more stable in micelles, SRP concentrations were lower in interstitial water, the contents of different
forms of phosphorus and the amounts of aerobic bacteria were lower while respiration efficiency was
higher in sediments. Phosphobacteria, both inorganic and organic and other microorganisms were
abundant in surface water but rare in sediments. Therefore, internal phosphorus may substantially flux
into water column by enzymatic hydrolysis and anaerobic release, together with mobility of bacteria,
thereby initiating the bloom. In short, biological mechanism may act in concert with physical and
chemical factors to drive the internal phosphorus release and accelerate lake eutrophication.
Keywords: dissolved phosphatase, reverse micelle, internal loading, phosphorus form of sediment, mi-
crobiology, eutrophication, Microcystis bloom.
1 Introduction
Eutrophication is one of the serious environmental
problems in the world with the symptom of excess
algal growth caused by the increase of nutrient input,
especially phosphorus. Internal phosphorus loading
was closely related to eutrophication, which may im-
pede the improvement of water quality even after de-
creasing the external loading
[1]
. Alkaline phosphatase
can catalyze organic phosphorus into orthophosphate
in water body and sediments. This is an important ap-
proach for the supply of bioavailable phosphorus
[2]
.
There must be a relation between alkaline phosphatase
and eutrophication
[3]
. Dissolved alkaline phosphatase
(DAP) may effectively accelerate the phosphorus cy-
cling due to the high proportion and continuity of
catalytic capability
[4,5]
. Based on the regulation of al-
kaline phosphatase synthesis, its secretion and stability,
Li et al. proposed the use of alkaline phosphatase ac-
Contributions of phosphatase and microbial activity to internal phosphorus loading 103

tivities for an assessment of the P-status
[6]
. However,
the relationship between DAP and internal phosphorus
loading as well as eutrophication has not been ad-
dressed. Microorganisms may regulate the abiotic ef-
fect in the process of phosphorus exchange of sedi-
ment-water interface due to the fluctuating redox con-
ditions in the sediment
[7]
. In addition, the microbial
community structure likely plays a major and direct
role in the release and uptake of phosphorus from the
sediment
[8]
. The decrease in potential for P release
from exposed sediments was caused by a shift in bac-
terial community structure
[9]
. Microorganisms can de-
compose organic phosphorus, store the polyphos-
phate
[10]
, and lower the redox potential by consuming
oxygen, thus enhancing the release of phosphorus
from the iron-P complexes
[11]
. Microorganisms play an
important role in the process of phosphorus cycling.
Lactate bacteria can resolve phosphate and make the
SRP concentration increase in the Dianchi Lake. The
enrichment of phosphorus caused by assemble phos-
phorus bacteria and the mineralization of organic
phosphorus after their death are two important ways
resulting in hydrated phosphate deposit in the lake
[12]
.
In addition, protease activity was closely related to
nitrogen mineralization and release. Phosphatase ac-
tivity was related to phosphorus decomposition in the
sediment of West Lake
[13]
and polluted degree indi-
cated by microbial characteristics in the sediment of
Victoria Bay of Hongkong
[14]
. Hence, the study of ac-
tivity and stability of dissolved phosphatase in benthic
layer of lakes, the characteristics of microbial commu-
nity, and phosphorus form of sediment would be help-
ful for understanding the mechanism of internal load-
ing cycling and the contribution of microbial commu-
nity to internal phosphorus loading in lakes.
DAP plays a crucial role in phosphorus cycling of
lakes. Its behavior varied in different lakes. In poly-
humic Lake Skjervatjern and Lake Mekkojarvi of
Germany, DAP accounted for 60%80% in total ac-
tivity
[15]
. It exhibited very low activity in turbid Aus-
tralian river
[16]
. In addition, the relation between DAP
and phosphorus is very complex. In oligotrophic Lake
Herrensee, DAP was responsible for more than half of
the total hydrolysis of organic phosphorus
[17]
, but
showed relatively low activity in Lake Lawrence with
the lack of phosphorus, USA
[18]
. The relative propor-
tion of DAP was independent of phosphorus in Ger-
man Lake Schohsee
[19]
. It may represent either a major
or an insignificant proportion of total phosphatase in
five Oklahoma lakes, depending on the limnological
characteristics of lake ecosystem
[20]
. Both zooplank-
ton
[21]
and bacteria
[22]
could support the growth of al-
gae by actively excreting dissolved phosphatase to
supply the bioavailable phosphorus in water, and ex-
tracellular substance produced by algae can provide
carbon source for bacteria. In short, dissolved phos-
phatase in lakes was diverse, which reflected the abil-
ity of plankton to adapt environments. The diversity of
DAP was shown in terms of its origin, catalytic effi-
ciency and temporal and spatial variations, which re-
fracted the complex ecological relations in lakes. Fur-
thermore, the cationic surfactants enhanced the acid
phosphatase activity, while anionic surfactants inhib-
ited the activity completely, and neutral surfactants
decreased the activity also, but had little or no effect
on kinetics of the enzyme. In unconventional medium,
both V
max
and K
m
values of phosphatase increased sig-
nificantly due to the slight folding of enzyme mole-
cule
[23]
. If we introduced unconventional medium
method into the research of freshwater ecology,
namely adding surfactants and organic solvents with
different proportions to lake water filtered through
0.45-m filters with higher DAP activity, we may give
further description of DAP and its stability in this re-
verse micelle system.
The reverse micelle is a transparent and thermody-
namic stability multi-phase organic system, which
consists of an aqueous micro-domain (water pool)
facing the polar heads of the surfactant that surrounds
this core interacting with the organic solvent, through
hydrophobic chains
[24]
. The water structure in the
aqueous core may resemble that of the water adjacent
to biological membranes, suggesting that this system
is a model of biological structure
[25]
. Thus, the enzy-
matic activity in the water pool of reverse micelle is
stable and may avoid the disturbance of microorgan-
isms.
This paper discussed the relationship between
phosphatase (especially activity and stability of dis-
solved phosphatase) and eutrophication in lakes with
different trophic levels (Lake Lianhua, Lake Longyang
and Lake Taihu) at spatial and vertical scales. The ob-
104 Science in China: Series D Earth Sciences
jectives of this study are to test the feasibility of ap-
plying micellar enzymology method to the research of
dissolved phosphatase in lakes and understand the
contribution of activity and stability of phosphatase as
well as microbial activity to internal loading and lake
eutrophication.

2 Materials and method
2.1 Lake situations and sampling sites
Lake Lianhua and Lake Longyang are situated in
Hanyang, Wuhan, China. Lake Longyang was a beau-
tiful shallow lake in the 1950s, and the area decreased
gradually due to increased wastewater discharges,
aquaculture and agriculture activities in the 1970s,
then reduced to 2/3 of the origin in the 1990s and lake
water was polluted severely. Lake Lianhua is a small
recreational lake, and water quality was improved
markedly due to the growth of submerged macro-
phytes. Lake Taihu is the third largest freshwater lake
in China, situated in the south of the Yangtze River
delta. Over the recent two decades, the lake has had a
deterioration of its water quality in many parts due to
increased anthropogenic inputs, especially in Lake
Wulihu, a large embayment of Lake Taihu, which is
relatively close with slow flowing velocity and long
water exchange period. The total area of Lake Wulihu
is 5.6 km
2
, with a length of 0.31.5 km (N-S), a
width of 6 km (E-W), and an average depth of about
1.95 m. Lake Wulihu is the most eutrophic area of
Lake Taihu. Therefore, dredging was started on a large
scale in 20032004. The basic data of studied lakes
are shown in Table 1. In our investigation, two sam-
pling sites were designed respectively in Lake Lianhua
and Lake Longyang, and six sampling sites were cho-
sen in field experiment in Lake Wulihu, including
Sites A(313224.6N 1201322.6E), C(31321.7N
1201318.7E), F(313158.1N 1201323.0E) and
E located in Lake Wulihu, and Sites B and D
(313132.3N 1201237.6E) located in Lake Taihu.
Sites A and F were dredged before January 2004, Site
C was dredged after January 2004, Sites B, D and E
were not dredged, and Site B Microcystis bloomed.
Sampling sites designs are shown in Fig. 1.
Table 1 Basic data of water quality in Lake Lianhua, Lake Longyang
and Lake Taihu (Samples of Lake Lianhua were collected on April 16,
2004; those of Lake Longyang were collected on May 10, 2004)
Sampling sites
Area
(km
2
)
Depth
(cm)
Transparency
(cm)
pH
Lake Lianhua 0.076 100 42 7.78
Lake Longyang 1.800 140 31 8.02
Lake Wulihu (Lake
Taihu)
5.600* 195* 50** 7.62*
* Data from ref. [26]; ** data from ref. [27].

Fig. 1. Sampling sites design and location of Lakes Lianhua, Long-
yang and Taihu.
2.2 Samples collection and preparation
Surface water of Lake Lianhua and Lake Longyang
was collected on April 16 and May 10, 2004, respec-
tively. Sediments were collected either by a Peterson
grab sampler, or by a hand-driven stainless steel corer
50 cm long with an internal diameter of 5.4 cm in
Lake Taihu on January 13 and September 27, 2004.
Interstitial water was extracted from the sediment by
Contributions of phosphatase and microbial activity to internal phosphorus loading 105

centrifugation at 3000 r/m for 30 minutes. The con-
centrations of soluble reactive phosphorus (SRP) and
chlorophyll a in surface water were measured after all
water samples were filtered through pre-washed
0.45-m filters
[28]
. Filtered water sample was concen-
trated to 40 mL with a rotary evaporator at 35 for
analysis of the DAP in the reverse micelles. All sam-
ples were stored in a refrigeratory.
2.3 Size-fractionation of alkaline phosphatase activ-
ity (APA)
All water samples were filtered through 0.45 and
3.0 m membrane filters. The contributions of APA to
the algal and bacterial fractions were calculated as
follows:
A = U F (3.0) and B = F (3.0) F (0.45),
where A = activity in algal fraction, i.e. in fraction
larger than 3.0 m, B = activity in bacterial fraction,
i.e. in fraction 0.453.0 m, U = activity of unfiltered
water sample, i.e. total APA, F (3.0) = activity in water
sample prefiltered through 3.0 m, and F (0.45) = ac-
tivity in water sample filtered through 0.45 m. The
final p-nitro-phenylphosphate (pNPP) concentration
(0.3 mmol L
1
) was used for the size-fractionation of
APA
[29]
.
2.4 DAP in reverse micelles
Each concentrated sample of 4.15 mL was injected
into a reverse micelle of 0.2 mol/L hexadecyltrime-
thylammonium bromide (CTAB) and 1 mol/L 1-
bytanol (as co-surfactant) in cyclehexane, and then
made this system clear by acute oscillation. The polar
cores of the micelles have the ability to solubilize a
significant amount of water and form the water pool.
The enzyme and substrate (pNPP) can interact with
each other in the water pool. The pH and water con-
tent W
0
was adjusted by 0.1 mol/L TrisHCl buffer
[30]
.
The reverse micelles with concentrated water samples
of Lake Lianhua and Lake Longyang were incubated
in different W
0
conditions (pH 8.5, 37), different pH
conditions (W
0
14, 37) and different temperature
conditions (pH 8.5, W
0
14). The reverse micelles with
concentrated water samples of Sites B and D in Lake
Taihu were incubated in W
0
14, pH 8.5, temperature
37 conditions . The samples in the reverse micelles
were collected in different incubation time. The reac-
tion was started by injection of the pNPP dissolved in
0.1 mol/L Tris-HCl buffer. The alkaline phosphatase
activity was determined by monitoring the increase in
absorbance at 400 nm after 4 h at 37 . T he molar
extinction coefficient in reverse micelles was 11300
mol/Lcm
1
. The concentration of the substrate with
the aqueous core of reverse micelles was constant at
18 mmol/L in all experiments mentioned above
[31]
.
2.5 Enumeration of different microbial functional
groups
Bacteria were enumerated using plate count tech-
niques for different functional groups. The numbers of
aerobic bacteria, organic and inorganic phosphobacte-
ria of surface, overlying water and sediment in Sites A,
C and D of Lake Taihu were determined by the serial
10-fold dilutions plate method. The culture medium
for aerobic bacteria: beef extract 3 g, peptone 5g, NaCl
5 g, agar 1518 g, distilled water 1000 mL. The cul-
ture medium for organic phosphobacteria: D-glucose
10g, (NH
4
)
2
SO
4
0.5g, MgSO
4
0.3g, NaCl 0.3g, KCl
0.3g, FeSO
4
0.036g, MnSO
4
0.03g, CaCO
3
5g, agar
1518g, distilled water 1000 mL. There needed 1mL
fresh vitellus as organic phosphorus source per 1215
mL culture medium; The culture medium for inorganic
phosphobacteria: D-glucose 10g, (NH
4
)
2
SO
4
0.5g,
MgSO
4
0.3g, NaCl 0.3g, KCl 0.3g, FeSO
4
0.036g,
MnSO
4
0.03g, Ca
3
(PO4)
2
2g, agar 1518g, distilled
water 1000 mL. Two replicate drops of 1 mL of three
dilutions sample were pipetted onto the plates. Pouring
plates started after cooling molten appropriate agar
medium for analysis of above three bacteria groups in
darkness to a temperature of 48 . Then dilution sa m-
ples and agar medium were mixed thoroughly and al-
lowed to cool in darkness for 2030 min. All plates
were incubated at 28 for 72 h. Colonies were
counted and the colony forming unit (CFU) ml/L or
g/L was calculated. Respiration efficiency of sediment
was determined by alkali absorbing method
[32]
.
2.6 Phosphorus form of sediment
Sediment phosphorus fractionation was carried out
according to Golterman, which grouped sediment P
into iron-bound P (Fe(OOH) P), calcium-bound P
106 Science in China: Series D Earth Sciences
phorus forms in surface sediment at the undredged site
(Site D) and SRP concentration in the interstitial water
(Sites B and D) were significantly higher than those at
the dredged sites (Sites A and C, Fig. 3; Table 2).
Chlorophyll a concentration showed the same trend
(Table 3). In addition, Microcystis sp. bloom occurred
at Site B then.
(CaCO
3
P), acid soluble organic P (ASOP) and hot
NaOH extractable residual organic P (NaOHP
extr
)
[33]
.
3 Results
3.1 Internal phosphorus loading and productivity
The concentrations of different phosphorus forms in
different depths of sediment at the undredged sites
(Sites E and C) were markedly higher than those at the
dredged sites (Sites A and F, Fig. 2) in January 2004.
In September, the concentrations of different phos-
3.2 Alkaline phosphatase activity and stability
Different size-fractionated APA in the interstitial
water was higher than those in the overlying water of

Fig. 2. The concentration of different form phosphorus at different depth of Lake Taihu (2004-01).

Table 2 The concentration of SRP in overlying and interstitial water of Lake Taihu (2004-09)
Sampling sites A B C D
Overlying
water
Interstitial
water

Overlying
water
Interstitial
water
Overlying
water
Interstitial
water

Overlying
water
Interstitial
water
SRP (mgL
1
)

0.018 0.019 0.040 0.022 0.024 0.017 0.022 0.126

Table 3 The concentration of chlorophyll a in surface water of Lake Taihu (2004-09)
Sampling sites A B C D
Surface water
Overlying
water
Surface
water
Overlying
water
Surface
water
Overlying
water

Surface
water
Overlying
water
Chlorophyll a (gL
1
)
0.056 32.55 12.68 1.67 11.72 13.02
, Undetectable.

Contributions of phosphatase and microbial activity to internal phosphorus loading 107

Fig. 3. The concentration of different form phosphorus at surface sedi-
ment of Lake Taihu (2004-09).

different sampling sites of Lake Taihu. Horizontally,
the APA of Site B where Microcystis sp. bloomed
showed the higher level, especially the dissolved parts
(Fig. 4).
The activity of DAP in the interstitial water of Site
B in reverse micelles began to decrease after 144 h
incubation, whereas the responding value was 72 h in
the surface and overlying water (Fig. 5). The activity
of DAP of Lake Longyang in micelles apparently ex-
hibited higher values than that in Lake Lianhua. The
latter decreased sharply at 48 h, and then dropped at
96 h again, whereas the former may retain relatively
high level for 192 h in different W
0
conditions (Fig.
6(a)). DAP of Lakes Longyang and Lianhua in mi-
celles was unstable at higher temperature. At 50, the
activity began to decrease after 35 h and 65 h incuba-
tion, respectively. At 20 and 37, the activity of
DAP of Lake Lianhua (65 h) began to decrease prior
to Lake Longyang (170 h) (Fig. 6(b)); In different pH
conditions, the activity of DAP of Lake Longyang in
micelles appeared to decline after 170 h incubation,
whereas the responding value in Lake Lianhua was 40
h (Fig. 6(c)). Accordingly, the stability of DAP in mi-
celles was not affected by pH but closely related to its
origin. Shortly, the eutrophic Lake Longyang showed
higher enzymatic activity and stability in micelles.
3.3 Bacterial abundance and respiration efficiency
Site D in Lake Taihu showed the highest abundance
of different functional bacterial groups in the surface,
overlying water and sediments among all sampling
sites (Table 4). In Site B, the bacterial abundances ex-
hibited relatively higher level in the surface water,
while aerobic bacterial abundance in sediment with
higher respiration efficiency was undetectable. Or-
ganic phosphobacteria and inorganic phosphobacteria
were enriched in sediments at all sampling sites (ex-
cept inorganic phosphobacteria at Site B).


Fig. 4. Size-fractionated activity of alkaline phosphatase in overlying and interstitial water of Lake Taihu (2004-09).

108 Science in China: Series D Earth Sciences

Fig. 5. The stability of dissolved alkaline phosphatase of surface, overlying and interstitial water at Sites B and D of Lake Taihu in reverse micelles
(W
0
:14; pH:8.5; T:37) (2004-09).

Table 4 The bacterial abundance and respiration efficiency in all sampling sites of Lake Taihu (2004-09)
Sampling sites
Aerobic bacteria
(10
3
CFU/mL; 10
3
CFU /g)
Organic phosphobacteria
(CFU /mL; CFU/g)
Inorganic phosphobacteria
(CFU /mL; CFU /g)
Respiration efficiency
(mLCO
2
/g.h)
Surface water of Site A 31 90
Overlying water of Site A 1.7 215
Sediment of Site A 1.9 30000 1500 2.46
Surface water of Site B 122 400 280
Overlying water of Site B 5.4 9
Sediment of Site B 1800 5.85
Surface water of Site C 3.1
Overlying water of Site C 2.7 230
Sediment of Site C 440 20500 1900 2.55
Surface water of Site D 2.8 385 410
Overlying water of Site D 144 1100 600
Sediment of Site D 1620 27000 2900 4.80
, Undetectable.

4 Discussion
Eutrophic symptoms, such as high productivity
(Sites B and D) and Microcystis sp. bloom (Site B) in
Lake Taihu, were closely related with internal load-
ings.
4.1 Internal loading and anaerobic release of
phosphorus
The concentrations of different phosphorus forms in
different depths of sediments at the undredged sites
were markedly higher than those at the dredged sites

Contributions of phosphatase and microbial activity to internal phosphorus loading 109

Fig. 6. The stability of dissolved alkaline phosphatase of Lake Longyang and Lake Lianhua in micelles. (a) Different W
0
conditions; (b) different
temperature conditions; (c) different pH conditions.

(Fig. 2) in January 2004. The lower redox potential in
sediments of the undredged sites was observed, for
example, the value was 102 mV in Site C, whereas
79 mV in Site A. In September, the concentration of
SRP in the interstitial water at Site B (with Microcystis
sp. bloomed) was considerably low (Table 2), and dif-

110 Science in China: Series D Earth Sciences

ferent phosphorus forms in surface sediment exhibited
a lower value than that at the undredged Site D, which
was approximate to the level at dredged Sites A and C
(Fig. 3). In addition, higher respiration efficiency and
lower aerobic bacteria abundance in sediments of Site
B indicated the anoxic status (Table 4). Hence, the
anaerobic release of internal phosphorus loading may
be an important reason for the occurrence of Micro-
cystis sp. bloom
4.2 High activity and stability of phosphatase
The APA in water column may act as an indicator
for the phosphorus status and eutrophication degree.
The APA exhibited higher level in eutrophic lakes
[34]

and gulfs
[35]
. Contrarily, the APA and phosphorus
concentration were low in lake water with the growth
of macrophytes
[36,37]
. Different size-fractionated APA
in Site B generally showed the highest level, espe-
cially the DAP in interstitial water (Fig. 4). Markedly
higher activity and stability of DAP in micelles and
SRP concentration in the interstitial water of Site D
were observed (Table 2, Fig. 5). Therefore, there was a
close relationship between phosphatase activity and
eutrophication, which may be supported by the data
from other lakes. In Lake Longyang, the SRP and
chlorophyll a concentrations were 0.418 mgL
1
and
24.41 gL
1
respectively, while in Lake Lianhua, the
responding values were 0.038 mgL
1
and 10.23
gL
1
. Thus Lake Longyang was more eutrophic rela-
tive to Lake Lianhua. In addition, its size-fractionated
APA was also higher. In Lake Longyang, APA associ-
ated with coarser (>3.0 m) and finer (0.453.0 m)
particles and DAP activities were 19.8, 6.2 and 35.5
nmolL
1
min
1
respectively, while in Lake Lianhua
the responding values were 12.1, 0.3 and 24.2
nmolL
1
min
1
. It means that the eutrophic lake
showed not only significantly higher APA with differ-
ent forms, but also significantly higher and more sta-
ble DAP activity in micelles (Fig. 6).
W
0
may regulate the micro-environment in water
pool of micelles and influence the enzymatic activity
and stability
[38]
. The thermal stability of enzyme de-
pends on the water content, decreasing when W
0
value
increases
[39]
, which was consistent with the results in
Lake Longyang (Fig. 6(a)). In lower water content, the
reversed micelles are capable of rearranging to ac-
commodate a protein with dimensions higher than the
water pool due to the possibility of rearrangement of
the surfactant chains. For example, at W
0
2.7 the mi-
celles have a radius of inner cavity of 8, while the
cutinase can be represented by a sphere with 21.6.
This implies that upon encapsulation, the micelles are
enlarged to host the protein. Cutinase in such small
micelles is structurally immobilized which leads to a
stabilization through a rigidification of its structure,
since its secondary and teriary structures are preserved
from denaturation usually associated to an unfolding
process
[40]
. Hence, low W
0
coupled with high stability
is related with enzyme molecule. However, at low W
0

the stability of DAP in Lake Lianhua decreased, sug-
gesting the difference of size of enzymatic molecule in
two lakes.
In three designed temperature conditions, DAP of
two lakes in micelles lost its activity completely in a
short time at 50 (Fig. 6(b)). The polyphenol oxidase
activity decreased to the lowest level at 70. The
possible explanation is that high temperature can
modify the physical characteristics of micelles, and
those changes probably include the rearrangement of
surfactant molecules influencing enzymatic conforma-
tion and activity
[41,42]
. In detail, solvation is necessary
for protein function. However, excessive solvation
may lead to the loss of the native protein structure.
This is more patent at high temperatures in which
many of the weak bonds that maintain the native
structure are destabilized and solvated
[30]
. In the same
temperature condition, the stability of DAP of two
lakes in micelles represented evidently distinct feature
(Fig. 6(b)), implying the difference of enzymatic con-
formation.
In general, the stability of phosphatase in micelles
cannot be affected by pH
[43]
, which was also proved in
our results (Fig. 6(c)). In the micellar microenviron-
ment, the ideas that pH of the initial buffer solution
changes upon micelle formation is widely spread, and
the ionogenic groups of the enzyme will be affected by
the microenvironment and their ionization state se-
verely influences the interaction with substrate and
inhibition by products
[44]
. For example, pH may acti-
vate Phosphatidylinositol-specific phospholipase by
disrupting ionogenic groups leading to a conforma-
tional change
[45]
. Furthermore, some changes are veri-
Contributions of phosphatase and microbial activity to internal phosphorus loading 111

fied to pH, but they depend on the amino residues of
each protein
[46]
. Hence, the variation of enzymatic
activity with pH in micelles was related to ionogenic
groups. In the same pH conditions, there was distinctly
different activity and stability of DAP in two lakes
(Fig. 6(c)), indicating the different amino residues of
enzyme.
Therefore, in the lakes and some hypolimnion with
higher trophic levels, DAP showed higher activity and
stability. The variations in DAP stability in micelle, as
affected by different W
0
, temperature and pH condi-
tions, reflected the characteristics of molecular size,
conformation and active residues of the enzyme, sug-
gesting the existence of isozymes in different lakes.
There existed phosphatase isozymes dependent on pH
in the streams
[47]
. Extracellular enzymes with different
origins in seawater may hydrolyze structurally distinct
polysaccharides by different velocities, implying that
microbial communities in different habitats can pro-
duce extracellular enzymes with specific substrates
and similar functions
[48]
. This paper provided further
evidence for the occurence of dissolved phosphatase
isozymes from the view-point of micellar enzymology.
In short, with higher activity and stability, DAP
isozymes could accelerate the eutrophication by
stimulating the release of internal phosphorus loading.
4.3 Microbial activity
Hypoxia may cause aerobic bacteria autolyze,
thereby resulting in cellular phosphorus release
[49]
,
which may occurred in the sediment of Site B. Thus,
the release of internal phosphorus was closely related
with anoxic status and the variation of microbial
community. In addition, among all the sampling sites,
Site B with Microcystis sp. bloom showed the most
abundant organic and inorganic phosphobacteria in the
surface water and the lowest amounts of bacteria in
sediments (Table 4), suggesting that bacteria in the
surface water may migrate from the sediment. A pos-
sible explanation is that organic carbon produced by
bloom algae induces the mobility of bacteria, which is
positively related with particle organic matter
[50]
. It is
supposed that there exists a metabolic coupling be-
tween bacteria and bloom algae. Bacteria provide in-
organic phosphorus through decomposition for algae
bloom, and in turn, the algae provide substantial or-
ganic carbon as bacterial carbon sources, reflecting a
mutuality between phytoplankton and bacteria, even
they compete with each other for inorganic phospho-
rus
[51]
. Hence, the mobility of bacteria coupled with
the phosphorus release should be an additional content
of internal loadings.
5 Conclusions
In Lake Taihu, the undredged zone had the symp-
toms of eutrophication with high phosphorus loading
and the releasing potential under anaerobic conditions
in sediments. The phosphatase activity was closely
related with the degree of eutrophication in the lake.
The behavior of DAP in reverse micelle may reflect its
characteristics of molecular size, conformation and
active residues. High and stable activity of DAP, com-
bined with the varying population of aerobic, inor-
ganic and organic phosphorus bacteria, substantially
contributed to the internal phosphorus loading and
promoted its flux. Biological mechanism may act in
concert with physical and chemical factors to facilitate
the internal phosphorus release and the excess growth
of algae, which may accelerate the process of lake eu-
trophication.
Acknowledgements The authors would like to thank Dr
Li Lin, Wu Zhongxing and Peng Liang for their help in
sampling. This work was supported by the Chinese Acad-
emy of Sciences (CAS) (Grant No. KZCX1-SW-12-II-
02-02), and the National Natural Basic Research Program of
China (Grant No.2002CB412304), the National Natural
Science Foundation of China (Grant No. 20177033). We are
also obliged for the funds (Grant No.2002AA601013).
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